Frataxin deficiency lowers lean mass and triggers the integrated stress response in skeletal muscle

Friedreich’s ataxia (FRDA) is an inherited disorder caused by reduced levels of frataxin (FXN), which is required for iron-sulfur cluster biogenesis. Neurological and cardiac comorbidities are prominent and have been a major focus of study. Skeletal muscle has received less attention despite indications that FXN loss affects it. Here, we show that lean mass is lower, whereas body mass index is unaltered, in separate cohorts of adults and children with FRDA. In adults, lower lean mass correlated with disease severity. To further investigate FXN loss in skeletal muscle, we used a transgenic mouse model of whole-body inducible and progressive FXN depletion. There was little impact of FXN loss when FXN was approximately 20% of control levels. When residual FXN was approximately 5% of control levels, muscle mass was lower along with absolute grip strength. When we examined mechanisms that can affect muscle mass, only global protein translation was lower, accompanied by integrated stress response (ISR) activation. Also in mice, aerobic exercise training, initiated prior to the muscle mass difference, improved running capacity, yet, muscle mass and the ISR remained as in untrained mice. Thus, FXN loss can lead to lower lean mass, with ISR activation, both of which are insensitive to exercise training.

Supplemental Figure 1: Exclusion tree diagram of participants for analysis, and lean mass in arms and legs in adults with FRDA and healthy controls.
(A) Description of how participants were excluded from the analysis. Of the 42 participants, 6 participated in two of the studies, and one participated in all three; in these cases, their most recent scans were analyzed. The remaining 34 participants with FRDA were 24 adults and 10 children.
All participants without FRDA were adults.
(B) Total lean mass in bilateral upper extremities and bilateral lower extremities was compared in adults with FRDA and healthy controls using two sample Wilcoxon rank-sum tests. The median values for total leg lean mass were nominally different in adults with FRDA (12.8kg, IQR 10.2-14.1kg) vs. healthy controls (13.5kg, IQR 12.5-18.3kg) (p=0.05). The median values for total arm lean mass were similar between the two groups (FRDA: 5.5, IQR 4.0-6.2 and healthy controls: 5.2, IQR 4.1-7.1) (p=0.47). corresponds to Ub-K48 and GRB2 immunoblots, which are shown in Figure 4C.   In all panels, individual data points are shown, and bars represent mean ± s.e.m. Statistical comparison was by unpaired t-test, *p<0.05, N.S.: not significant.

Mouse studies
Grip strength test. Grip strength was measured using the DFIS-2 Series Digital Force Gauge (Columbus Instruments, OH), as described (70). Grip strength testing was conducted by allowing the mouse to grasp a triangular metal bar that is attached to the force gauge. During the testing session, the mouse was allowed to grab the metal bar with both forepaws and then was quickly pulled away from gauge instrument, until the forelimbs released the bar. This provides a measurement of the force of grip strength. A similar procedure was used using mouse hindlimbs.
During each test, six measurements were recorded for each forelimb and hindlimb, using an interval of at least 60 s between each measurement. Mice were acclimated for two weeks (with two acclimation sessions per week) before the testing session.
Treadmill training protocol. Treadmill running was performed using an Exer3/6 Treadmill For the flat treadmill protocol, WT and TG were acclimated for 3 days. An experimental run was performed two days later, where the treadmill speed was increased progressively. The speed was increased from 0 m/min to 5 m/min (20 s of speed transition) and was maintained at 5 m/min for 720 s. From here, the speed was increased subsequently (60 s of transition for each speed) to 7.5 m/min (duration of 720 s) and then to 10 m/min (for 720 s). After this phase, the speed was progressively increased (60 s of transition for each speed) to 12.5, 15 and 17.5 m/min (600 s of duration for each period). Then, the treadmill speed was increased (60 s of transition) to 20 and 22.5 m/min (duration of 360 s for each speed). Finally, the speed was increased to 25 m/min (60 s of transition) and was maintained at this speed for 240s. The total running time was 90 min.
Isolation of skeletal muscle mitochondria. Skeletal muscle mitochondria were isolated following the protocol described in (71) (g)). The tissue was homogenized using a glass Potter-Elvehjem homogenizer with a Teflon pestle (500 r.p.m., 15 passes). After that, the tissue suspension was centrifuged at 2,000 r.p.m. for 10 min and the supernatant was collected and spun at 9,000 r.p.m. for 8 min. The pellet obtained was resuspended and incubated in BM for 5 min. Samples were then centrifuged at 2,000 r.p.m. for 10 min and supernatant obtained was filtered through a 70 µM cell strainer (Falcon 352250) and spun at 10,000 r.p.m. Finally, the pellet was resuspended in BM and its protein concentration was determined by bicinchoninic acid (BCA) assay (ThermoFisher Scientific 23225).